Redefining The Role Of Myosin Essential Light Chain In Cardiac Muscle

重新定义肌球蛋白必需轻链在心肌中的作用

• 批准号: 10376748
• 负责人: Danuta Szczesna-Cordary
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-05 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376748
• 关键词: Actins; Age; Alkalies; Amino Acids; Binding; Biological Assay; Calcium; Cardiac; Cardiac Muscle Contraction; Cardiomyopathies; Cells; Complex; Consumption; Data; Disease; Echocardiography; Etiology; Event; Fluorescence; Fluorescent Probes; Frequencies; Genes; Glycine; Head; Health; Heart; Heart Diseases; Homeostasis; Human; Hypertrophic Cardiomyopathy; Hypertrophy; In Vitro; Investigation; Label; Light; Link; Lysine; Measurement; Metabolic; Mitochondria; Modeling; Modification; Molecular; Motor; Mus; Muscle; Muscle Contraction; Muscle Fibers; Mutate; Mutation; Myocardium; Myosin ATPase; Myosin Alkali Light Chains; N-terminal; Organ; Organelles; Output; Oxidative Phosphorylation; Pathologic; Peroxisome Proliferator-Activated Receptors; Phenotype; Physiological; Play; Production; Proteins; Quantum Dots; RNA Splicing; Regulation; Respiration; Respiratory Chain; Role; Skeletal Muscle; Skin; Smooth Muscle Myosins; System; Testing; Thick Filament; Transgenic Mice; Transgenic Organisms; Variant; Ventricular; Work; base; cardiac muscle disease; cell motility; hemodynamics; hexokinase; in vivo; insight; mouse model; mutant; novel; novel therapeutics; papillary muscle; respiratory; response; sex

ABSTRACT The scientific premise of this application regards the cardiac specific expression of the long ELC containing the N-terminal ELC extension (N-ELC) and its physiologically important role in regulating myosin motor function and force production in muscle. We hypothesize that cardiac N-ELC works as a molecular linker and/or energetic switch of the actin-myosin interaction by regulating the transition of myosin cross-bridges from a super- relaxed (SRX) to a disordered-relaxed (DRX) state, and the proportion of myosin heads that are available for the interaction with actin. In DRX, the myosin heads protrude into the interfilament space but are restricted from binding to actin, while in the SRX state, the heads are neatly ordered along the thick filament axis but cycle at a highly inhibited ATP turnover rate. We further hypothesize that hypertrophic cardiomyopathy mutations in myosin ELC promote the SRX-to-DRX transition and the deletion of the N-ELC counteracts this transition and stabilizes the energy conservation SRX state. We will test these hypotheses using two mouse models of hypertrophic HCM-A57G (Alanine57Glycine) and restrictive RCM-E143K (Glutamate143Lysine) cardiomyopathy. We will also use transgenic Δ43 mice expressing a 43-aa truncated ELC, and double transgenic mice: A57GxΔ43 and E143KxΔ43, expressing Δ43-ELC in the background of HCM or RCM mutations. The data will be compared to age and sex matched WT-ELC mice expressing the human ventricular ELC (MYL3 gene). SPECIFIC AIM 1: DEFINE THE ROLE OF MYOSIN ELC AND ITS N-TERMINUS (N-ELC) IN THE REGULATION OF MYOSIN MOTOR FUNCTION AND CARDIAC MUSCLE CONTRACTION. This aim will focus on the molecular triggers of ELC-induced heart remodeling studied at the level of myosin molecules, myofibers and the whole heart. The N-ELC-dependent regulation of actin-myosin interactions will be studied using: 1) mant-ATP-chase assays and the proportion of myosin heads in SRX vs. DRX states; 2) Quantum dot-labeled actin±Tm-Tn motility assays; 3) Skinned and intact muscle fibers and force-pCa as well as force and calcium transient measurements; and 4) echocardiography and invasive hemodynamics to assess contractile responses of the heart in vivo. SPECIFIC AIM 2: THE ROLE MYOSIN ELC IN ENERGETIC REMODELING OF THE HEART IN MOUSE MODELS OF CARDIOMYOPATHY. The analysis of the metabolic landscape in A57G, E143K, Δ43, A57GxΔ43 and E143KxΔ43 vs. WT hearts will be performed to identify the specific mechanisms that govern energy utilization in all ELC-mutant vs. WT mice. Mitochondrial function will be assessed by determination of mitochondrial respiration, enzymatic activity of respiratory complexes as well as ATP production/consumption. Steady-state levels of candidate proteins implicated in energy production such as Hexokinases I and II, various mitochondrial respiratory chain subunits (Complexes I to V) as well as peroxisome proliferator-activated receptor coactivator α (PGC-1α) will be assessed in all models to characterize the broader implications of cardiac muscle disease resulting from ELC-linked modifications.

抽象的 本申请的科学前提是含有长ELC的心脏特异性表达 N 端 ELC 延伸 (N-ELC) 及其在调节肌球蛋白运动功能中的重要生理作用 和肌肉力量的产生。我们假设心脏 N-ELC 作为分子连接器和/或 通过调节肌球蛋白跨桥从超级-肌球蛋白相互作用的能量转换 放松（SRX）状态到无序放松（DRX）状态，以及可用于该状态的肌球蛋白头的比例 与肌动蛋白的相互作用。在 DRX 中，肌球蛋白头伸入丝间空间，但受到限制 与肌动蛋白结合，而在 SRX 状态下，头部沿着粗丝轴整齐排列，但以 高度抑制 ATP 周转率。我们进一步假设肥厚型心肌病突变 肌球蛋白 ELC 促进 SRX 到 DRX 的转变，而 N-ELC 的删除会抵消这种转变， 稳定能量守恒SRX状态。我们将使用两种小鼠模型来测试这些假设 肥厚型 HCM-A57G（丙氨酸 57甘氨酸）和限制性 RCM-E143K（谷氨酸 143赖氨酸） 心肌病。我们还将使用表达 43-aa 截短 ELC 的转基因 Δ43 小鼠，以及双转基因 小鼠：A57GxΔ43 和 E143KxΔ43，在 HCM 或 RCM 突变背景下表达 Δ43-ELC。数据 将与年龄和性别匹配的表达人心室 ELC（MYL3 基因）的 WT-ELC 小鼠进行比较。 具体目标 1：定义肌球蛋白 ELC 及其 N 末端 (N-ELC) 在法规中的作用 肌球蛋白运动功能和心肌收缩的影响。这一目标将集中在分子 在肌球蛋白分子、肌纤维和整体水平上研究 ELC 诱导的心脏重塑的触发因素 心。将使用以下方法研究肌动蛋白-肌球蛋白相互作用的 N-ELC 依赖性调节：1) mant-ATP-chase SRX 与 DRX 状态下的测定和肌球蛋白头的比例； 2) 量子点标记的肌动蛋白±Tm-Tn 运动性 化验； 3) 去皮和完整的肌纤维和力-pCa以及力和钙瞬态测量； 4）超声心动图和侵入性血流动力学，以评估体内心脏的收缩反应。 具体目标 2：肌球蛋白 ELC 在小鼠模型心脏能量重塑中的作用 心肌病。 A57G、E143K、Δ43、A57GxΔ43 和 A57GxΔ43 中代谢景观的分析 将进行 E143KxΔ43 与 WT 心脏对比，以确定控制能量利用的具体机制 在所有 ELC 突变小鼠与 WT 小鼠中。线粒体功能将通过测定线粒体来评估 呼吸作用、呼吸复合物的酶活性以及 ATP 的产生/消耗。稳定状态 与能量产生有关的候选蛋白质的水平，例如己糖激酶 I 和 II、各种线粒体 呼吸链亚基（复合物 I 至 V）以及过氧化物酶体增殖物激活受体共激活物 α (PGC-1α) 将在所有模型中进行评估，以表征心肌疾病的更广泛影响 由 ELC 连接修饰产生。